Technical Field
The presently disclosed invention embodiments relate generally to anti-FZD10 antibodies and to methods of using anti-FZD10 antibodies. In particular, the anti-FZD10 antibodies and the methods described herein are useful for the treatment of diseases associated with expression (typically overexpression) of FZD10, such as a variety of cancers, and for the identification of treatment regimens that comprise administration of anti-FZD10 antibodies alone or in combination with other agents. In addition, the methods are useful for regulating the growth and differentiation of stem cells, such as embryonic, pluripotent, and cancer stem cells, with therapeutic implications in tissue regeneration and transplantation as well as cancer.
Description of the Related Art
Frizzled antigens are a family of G protein coupled receptor-like cell surface receptors that mediate biological signal transduction and that have binding sites for Wnt protein ligands, which are secreted molecules that act as selective upregulators of specific gene expression. Members of the Frizzled and Wnt families of receptor-ligand pairs regulate embryonic development, and may also play a role in cellular proliferation and in determining the ultimate fate of cells during embryogenesis. Most frizzled receptors are functionally coupled to the β-catenin or “canonical” Wnt signaling pathway, in which Wnt ligand binding to a cell surface frizzled receptor leads sequentially to the activation of cytoplasmic dishevelled proteins, inhibition of intracellular GSK-3 kinase, nuclear accumulation of β-catenin and, through interaction of β-catenin in the nucleus with TCF or LEF transcription factors, transcriptional activation of Wnt target genes. Other Wnt signaling pathways involving protein kinase C (PKC) and calcium flux have been described for some Frizzled and Wnt family members, but it is not yet clear if these all represent distinct pathways or if any one or more of them may be integrated with the canonical Wnt signaling pathway.
There are 19 Wnt and 10 Frizzled (Fz or FZD) genes identified thus far in the human genome database (Venter et al., Science 291:1304-1351 (2001)). There are also five secreted Frizzled forms. Each Fz gene encodes an integral membrane protein with a large N-terminal extracellular portion, seven putative transmembrane domains, and a cytoplasmic tail (Wang et al., J. Biol. Chem. 271, 4468-4476(1996); Vinson et al., Nature (London) 338, 263-264(1989)). Near the NH2 terminus of the extracellular portion is a cysteine-rich domain (CRD) that is well conserved among other members of the FZ family (Wang et al., J. Biol. Chem. 271, 4468-4476(1996)). The CRD, comprised of 110 amino acid residues, including ten invariant cysteines, is the putative binding site for Wnt ligands (Bhanot et al., Nature (London) 382, 225-230(1996). Frizzled receptors can dimerize in the cell membrane, and dimerization is correlated with activation of the Wnt/β-catenin pathway (Carron et al., Journal of Cell Science, 116:2541-2550 (2003)).
A human Fz gene family member, Frizzled-10 (FZD10), has been cloned and characterized (Koike et al., Biochem Biophys Res Commun. 262(1):39-43(1999)). Analysis of the FZD10 nucleotide sequence showed that the human FZD10 gene encodes a seven-transmembrane-receptor of 581 amino acids, including an amino-terminal cysteine-rich domain and a carboxy-terminal Ser/Thr-Xxx-Val motif. FZD10-encoding mRNA (4.0 kb) was detected in placenta, fetal kidney, fetal lung and brain. In adult brain, FZD10 mRNA was abundant in the cerebellum. The FZD10 gene was mapped to human chromosome 12q24.33. FZD10 shares 65.7% amino-acid identity with Frizzled-9 (FZD9). FZD10 and FZD9 constitute a subfamily among the Frizzled genes. FZD10 is the receptor for the Wnt ligand proteins WNT7a and WNT7b. There is 93% identity between mouse and human FZD10. A human FZD10 (also known as FZ10, CD350, FzE7, hFZ10, frizzled homologue 10, FZ-10) amino acid sequence is set forth in SEQ ID NO:28.
In normal tissues, expression levels of the FZD10 protein are very low or absent (e.g., not detectable by conventional means) in vital organs, and present at low levels in superficial mucosa of the stomach and colon, in kidney proximal and distal tubules, in endometrial stroma and in placenta. Readily detectable FZD10 expression, however, has been shown in various cancers, such as synovial sarcoma (92%; Nagayama et al., 2002 Cancer Res. 62:5859), gastric carcinoma (40%; Kirikoshi et al., 2001 Int. J. Oncol. 19:767) and colorectal carcinoma (25%; Nagayama et al., 2009 Cancer Sci. 100:405). Specific siRNA knockdown of FZD10 expression resulted in synovial sarcoma cell growth inhibition in vitro, and polyclonal anti-FZD10 antibodies were shown to mediate as antibody dependent cell-mediated cytotoxicity (ADCC) against FZD10-overexpressing synovial sarcoma cells in vitro, and to inhibit synovial sarcoma xenograft tumor growth in vivo. (Nagayama et al., 2005 Oncogene 24:6201). A radiolabeled anti-FZD10 monoclonal antibody was internalized by antigen-bearing tumor cells and dramatically suppressed synovial sarcoma xenograft tumor growth in vivo. (Fukukawa et al., 2008 Cancer Sci. 99:437).
Wnt-FZD pathways are thus activated in many cancers, yet the FZD receptors have yet to be effectively developed as therapeutic targets. Clearly there is a need for anti-FZD10 monoclonal antibodies that are amenable to clinical development. The presently described invention embodiments address this need and offer other related advantages.